After motoneurons (MNs) lose functional connectivity with muscle by axotomy, a variety of changes occur in MN properties including a switch to a regenerative mode. These changes return to normal when motor axons re-establish synaptic contact with muscle. This evidence indicates that synaptic contact mediates important interactions between muscle and MNs that normally enable expression of normal MN properties and inhibit regeneration, but underlying mechanisms are poorly understood. We have shown that blockade of motor endplate acetylcholine receptors (ACHRs) produces axotomy-like changes in MN current threshold for spike activation (rheobase current), an important metric of MN excitability. Additional evidence we have obtained shows that muscle fiber action potential or mechanical activity is not involved in this signaling. These observations suggest that retrograde signaling between muscle and MNs may be accomplished via ACHR activation. An important question is whether loss of ACHR-mediated signaling can provoke a wider range of post-axotomy effects. If so, then the effects of axotomy may be based on the loss of ACHR-mediated retrograde signaling from muscle rather than injury itself. This novel idea will be tested in Specific Aim 1. Other available evidence indicates that a significant Ca2+ influx is initiated by activation of endplate ACHRs. Such currents could serve to activate downstream mechanisms within muscle and thus link ACHR activation to eventual production of retrograde signals to MNs. Included among several Ca2+-sensitive molecules located at the motor endplate is neuronal nitric oxide synthase (nNOS). Ca2+ activation of nNOS produces nitric oxide (NO) which may signal MNs directly via diffusion to motor terminals or activate further downstream cascades at the motor endplate that ultimately provide retrograde signaling. In Specific Aim 2, we will test the involvement of nNOS in retrograde signaling to MNs by determining whether exogenous NO can prevent axotomy-like changes in MN properties after ACHR blockade. The results of these studies will add new insight into factors that trigger the axotomy response in MNs and begin the identification of molecular mechanisms in muscle which underlie signaling that controls at a minimum MN excitability. PUBLIC HEALTH RELEVANCE: This work is focused on understanding mechanisms underlying retrograde signaling between muscle and motor neurons. Based on data we collected, we have developed the hypothesis that this signaling is initiated by motor endplate acetylcholine receptor activation. The purpose of the proposed studies is to test whether loss of this signaling underlies the motor neuron response to axotomy and to begin identification of molecular mechanisms in muscle that mediate retrograde signaling.